Voltage-gated sodium channels (NAChs) are vital for electrical signaling and conduction in the nervous system. There are at least ten NaCh genes in rodents, and each gene has a known ortholog in humans. The specific roles of each NaCh subtype are unknown. One hypothesis is that subtypes are differentially distributed and modulated. This proposal is focused upon the molecular basis for modulation and localization of brain NaCh subtypes. The goal of Aim 1 is to identify proteins that bind to the cytoplasmic domains of brain NaChs (esp. Nav1.6) and are responsible for (a) modulation of channel fiinction and (b) targeting to different subcellular sites. Two complementary methods are being used to find and isolate proteins associated with NaChs: (1) the yeast two-hybrid assay and (2) protein purification/mass spectrometry. Aim 2 is focused upon the interactions of NaChs with calmodulin, which was isolated with the yeast two-hybrid assay. This interaction with brain NaChs will be characterized biochemically and electrophysiologically. Aim 3 utilizes imaging techniques to study the subcellular distribution of brain sodium channels (esp., Nav 1.6) and their binding proteins. After characterizing this distribution, the effects of mutations in NaChs or in the binding proteins identified in Aim I will be studied. This research has both basic science and clinical relevance. Ion channels exist in complexes with other membrane, extracellular, and intracellular proteins. To understand the behavior of these channels, it is important to know which proteins are present in these complexes and hew the proteins interact with the channel. Mutations in muscle NaChs are responsible for some disorders of skeletal muscle and for long QT syndrome in cardiac muscle. It is expected that mutations in brain sodium channels and in the proteins that bind to NaChs will produce CNS disorders in humans, and the proposed studies will contribute to our understanding of these disorders.